Melanocortin-4 receptor (MC4R) is a G-protein-Coupled Receptor (GPCR) expressed in many areas of the brain including the paraventricular nucleus of the hypothalamus. At this location, MC4R expressing neurons receive signals generated by other neurons that release the orexigenic hormone alpha-MSH, which binds and activates MC4R. MC4R mutations account for most of the monogenetic defects that cause obesity, indicating a role in appetite control. Preliminary data from the PI show that MC4R functions as an atypical GPCR because, rather than being endocytosed in response to agonist stimulation, the receptor is constitutively internalized and recycled back to the plasma membrane. In addition it is found that alpha-MSH sequesters MC4R from the plasma membrane by blocking the receptor in an intracellular localization, rather than by increasing its rate of endocytosis. Preliminary results from the PI also indicate that several variants linked to human obesity have defective traffic along the biosynthetic pathway and are retained in the endoplasmic reticulum (ER). The central hypothesis of this proposal is that MC4R cell traffic is essential to modulate receptor activity. In Aim 1, by using a combination of fluorescence-based and biochemical assays, it is determined whether agonist-dependent post-translational modifications of MC4R and binding to specific factors control recycling of the receptor back to the plasma membrane and receptor re-sensitization. Immunoelectron microscopy will be used to determine the intracellular distribution of endogenous MC4R in neurons of the paraventricular nucleus of rat hypothalamus. In Aim 2 the hypothesis is tested that obesity-linked variants that are inefficiently expressed at the plasma membrane are retained in the ER as misfolded proteins, self-associate, and are inefficiently degraded. Possible routes to rescue cell surface expression of such variants will be investigated. It is anticipated that these studies will broaden our understanding of traffic and signaling of MC4R and of obesity- linked variants and will help find new targets for therapies against obesity. PUBLIC HEALTH RELEVANCE: In the last 10 years it has become clear that appetite and energy expenditure are controlled at a central level and that MC4R, a member of the GPCR family of proteins, plays an essential role in these pathways. The mechanisms of MC4R desensitization have not been studied in detail and here we propose to fill this knowledge gap. Defects in the mechanism by which other GPCR are desensitized appear to be important in the pathogenesis of many diseases including Parkinson's disease, mood disorders, and schizophrenia. Pharmacological strategies aimed at targeting the GPCR desensitization machinery including GRK and [unreadable]-arrestin are currently considered to treat brain disorder. On the basis of the mechanism by which MC4R desensitization occurs, similar pharmacological approaches may be considered for the treatment or prevention of obesity. In addition, we propose to understand the molecular mechanism by which specific human MC4R variants cause obesity and to identify novel factors involved in MC4R traffic and signaling. In conclusion, we anticipate that this project will help understand the cell biology of MC4R and that this will be important to find new therapeutic targets to prevent or reverse obesity.